This application is the National Stage of International Application No. PCT/EP99/03490, filed May 21, 1999, which claims priority to German Application No. 198 25 447.4, filed Jun. 6, 1998.
The present invention relates to insulin analogs which have an increased zinc binding ability, and to stable zinc complexes thereof which, in comparison with human insulin, have a delayed profile of action, to a process for their preparation and to their use, in particular in pharmaceutical preparations for the therapy of diabetes mellitus of type I and also type II.
Worldwide, approximately 120 million people suffer from diabetes mellitus. Among these, approximately 12 million are type I diabetics, for whom the substitution of the lacking endocrine insulin secretion is the only possible therapy at present. Those affected are prescribed insulin injections, as a rule several times daily, for life. Unlike type I diabetes, in type II diabetes there is not fundamentally a lack of insulin, but in a large number of cases, especially in the advanced stage, treatment with insulin, if appropriate in combination with an oral antidiabetic, is regarded as the most favorable form of therapy.
In healthy people, the release of insulin by the pancreas is strictly coupled to the concentration of the blood glucose. Increased blood glucose levels, such as occur after meals, are rapidly compensated by a corresponding increase in insulin secretion. In the fasting state, the plasma insulin level drops to a basal value which is sufficient to guarantee a continuous supply of insulin-sensitive organs and tissues with glucose and to keep the hepatic glucose production low during the night. The replacement of the endogenous insulin secretion by exogenous, mostly subcutaneous, administration of insulin as a rule does not nearly achieve the quality of the physiological regulation of the blood glucose described above. Frequently, there are losses of control of the blood glucose upward or downward, which in their most severe forms can be life-threatening. In addition, blood glucose levels which have been raised for years without initial symptoms, however, also represent a considerable risk to health. The large-scale DCCT study in the USA (The Diabetes Control and Complications Trial Research Group (1993) N, Engl. J. Med. 329, 977-986) clearly demonstrated that chronically raised blood glucose levels are largely responsible for the development of diabetic late damage. Diabetic late damage is micro- and macrovascular damage which is manifested, under certain circumstances, as retinopathy, nephropathy, or neuropathy and leads to blindness, kidney failure and the loss of extremities and is moreover accompanied by a high risk of cardiovascular diseases. It can be derived from this that an improved therapy of diabetes must primarily aim to keep the blood glucose as closely as possible in the physiological range. According to the concept of intensified insulin therapy, this should be achieved by a number of daily injections of rapid- and slow-acting insulin preparations. Rapid-acting formulations are given at mealtimes in order to level out the post-prandial increase in blood glucose. Slow-acting basal insulins should ensure the basic supply of insulin, in particular during the night, without leading to hypoglycemia.
The basal insulins available at present fulfill this requirement only inadequately. The frequently used NPH insulins especially have a too strongly pronounced maximum action and have too short an overall action. In the case of administration in the evening, this involves the risk of nightly hypoglycemia and morning hyperglycemia.
EP 0 821 006 discloses insulin analogs having increased zinc binding ability, which in combination with zinc have a delayed profile of action compared with human insulin. These analogs differ from human insulin essentially by variation of the amino acid in position A21 of the A chain and by addition of a histidine residue or of a peptide having 2 to 35 amino acid residues, which contains 1 to 5 histidine residues, in position B30 of the B chain.
It is the object of the present invention to provide further insulin analogs (analogs of human or animal insulin) which have an increased zinc binding ability, form a stable complex comprising a hexamer of the insulin analog and zinc, and, in a suitable preparation, make possible an improved therapy of diabetes mellitus of type I and of type II on subcutaneous injection as a result of the profile of action, which is delayed in comparison with human insulin.
Insulin analogs are derived from naturally occurring insulins, namely human insulin (see SEQ ID NO: 1: A chain of human insulin and SEQ ID NO: 2: B chain of human insulin) or animal insulins by substitution or absence of at least one naturally occurring amino acid residue and/or addition of at least one amino acid residue to the A and/or B chain of the naturally occurring insulin.
1. An insulin analog or a physiologically tolerable salt thereof of the formula I 
in which
(A1-A5) are the amino acid residues in the positions A1 to A5 of the A chain of human insulin (cf. SEQ ID NO: 1) or animal insulin,
(A15-A19) are the amino acid residues in the positions A15 to A19 of the A chain of human insulin (cf. SEQ ID NO: 1) or animal insulin,
(B8-B18) are the amino acid residues in the positions B8 to B18 of the B chain of human insulin (cf. SEQ ID NO: 2) or animal insulin,
(B20-B29) are the amino acid residues in the positions B20 to B29 of the B chain of human insulin (cf. SEQ ID NO: 2) or animal insulin,
R8 is Thr or Ala,
R9 is Ser or Gly,
R10 is lle or Val,
R14 is Tyr, His, Asp or Glu,
R21 is Asn, Asp, Gly, Ser, Thr, Ala, Glu or Gln,
R1 is any desired genetically encodable amino acid residue, absent or a hydrogen atom,
R2 is Val, Ala or Gly,
R3 is Asn, His, Glu or Asp,
R4 is Ala, Ser, Thr, Asn, Asp, Gln, Gly or Glu,
R30 is any desired genetically encodable amino acid residue or xe2x80x94OH,
Z is a hydrogen atom or a peptide residue having 1 to 4 genetically encodable amino acid residues, comprising 1 to 4 histidine residues (His),
with the proviso that at least one of the following is true: (1) when Z is a hydrogen atom, R1 or R3 is chosen from His, Glu, and Asp; (2) when R1 is a neutral or negatively charged amino acid residue, R3 is His; or (3) when Z is a hydrogen atom, R14 is chosen from His, Asp and Glu; and with the further proviso that when in formula I R3, R3 in combination with R21, or R3 in combination with R4 differs from human insulin, the insulin analog or the physiologically tolerable salt thereof of the formula I contains at least one additional variation from human insulin. (cf. SEQ ID NO: 1 and SEQ ID NO: 2).
Preferably, the insulin analog or the physiologically tolerable salt thereof is one wherein
2. R8 is Thr, R9 is Ser and R10 is lle,
3. R1 is Phe, His, Asn, Asp or Gly,
4. R30 is Thr, Ala or Ser or
5. wherein R21 is Asn and R1 is Phe.
6. A preferred embodiment of the present invention is an insulin analog or a physiologically tolerable salt thereof of the formula 1, wherein R2 is Val, R3 is Asn and R4 is Gln.
An insulin analog or a physiologically tolerable salt thereof of the formula I is furthermore preferred which is distinguished in that R14 is
7. Tyr,
8. His,
9. Asp or
10. Glu.
An insulin analog or a physiologically tolerable salt thereof of the formula I is furthermore preferred which is distinguished in that R30 is
11. Thr,
12. Ala,
13. Ser or
14. xe2x80x94OH.
An insulin analog or a physiologically tolerable salt thereof of the formula I is furthermore preferred which is distinguished in that Z is
15. His,
16. His-Ala- or
17. His-Ala-Ala-.
Examples of insulin analogs according to the present invention are
18. an insulin analog or a physiologically tolerable salt thereof of the formula I, which is distinguished in that the B chain has the sequence
His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys
(SEQ ID NO: 3), for example His(B0), des(B30) human insulin,
19. an insulin analog or a physiologically tolerable salt thereof of the formula I, which is distinguished in that the B chain has the sequence
His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
(SEQ ID NO: 4), for example His(B0)-human insulin,
20. an insulin analog or a physiologically tolerable salt thereof of the formula I, which is distinguished in that the B chain has the sequence
His Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
(SEQ ID NO: 5), for example His(B-1), Ala(B0) human insulin or
21. an insulin analog or a physiologically tolerable salt thereof of the formula I, which is distinguished in that the B chain has the sequence
His Ala Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
(SEQ ID NO: 6), for example His(B-2), Ala(B-1), Ala(B0)-human insulin.
The present invention furthermore relates to a process for the preparation of the insulin analog or of a physiologically tolerable salt thereof according to the present invention, comprising the construction of a replicable expression vehicle which contains a DNA sequence which codes for a precursor of the insulin analog having the amino acid sequence of formula II
Met-X2m-(Arg)p-Z-R1-R2-R3-R4-His-Leu-Cys-(B8-B18)-Cys-(B20-B29)-R30-X1n-Arg-(A1-A5)-Cys-Cys-R8-R9-R10-Cys-Ser-Leu-R14-(A15-A19)-Cys-R21xe2x80x83xe2x80x83II
in which
X1n is a peptide chain having n amino acid residues, where n is an integer from 0 to 34,
X2m is a peptide chain having m amino acid residues, where m is an integer from 0 to 20,
p is 0, 1 or 2,
R30 is any desired genetically encodable amino acid residue or is absent and
Z is absent or is a peptide residue having 1 to 4 genetically encodable amino acid residues, comprising 1 to 4 histidine residues (His)
and the other variables have the meanings mentioned above under No. 1, where the abovementioned provisos also apply, expression in a host cell and release of the insulin analog from its precursor using chemical and/or enzymatic methods.
The host cell is preferably a bacterium, particularly preferably the bacterium E. coli. 
The host cell is preferably a yeast, particularly preferably Saccharomyces cerevisiae. 
During expression in E. coli, the fusion proteins mentioned (SEQ ID NO: 7 to 9) as a rule form insoluble inclusion bodies, which can be isolated by centrifugation after cell disruption and are dissolved again using chaotropic additives (e.g. 8 M urea or 6 M guanidinium chloride). The dissolved fusion protein can be subjected to sulfitolysis, in which SH radicals are converted into S-sulfonates (e.g. R. C. Marshall and A. S. Iglis in, Practical Protein Chemistryxe2x80x94A Handbookxe2x80x99, edited by A. Darbre (1986), pages 49-53). The solubility of the fusion protein is thereby improved and purification, for example by means of anion-exchange or gel permeation chromatography, is facilitated.
The conversion of the derivatized fusion protein into preproinsulin with a native spatial structure and correctly formed disulfide bridges (folding) is carried out in dilute aqueous solution by addition of a limited amount of an SH reagent such as mercaptoethanol, cysteine or glutathione and subsequent aerial oxidation. Alternatively, the dissolved, underivatized fusion protein can also be directly folded under similar conditions (EP-A-0 600 372; EP-A-0 668 292).
Preproinsulin is then converted into biologically active insulin by limited proteolytic cleavage. For this, it is possible to use trypsin which removes the presequence indicated in formula II by Met-X2m-(Arg)p and cleaves at the peptide chain indicated by X1n-Arg and thus separates the B and A chain. As a rule, the sequence X1 begins with Arg, Arg2 or it is not present (n=0), so that after the cleavage an insulin derivative is present which is prolonged by Arg or Arg2 at the C terminus of the B chain. These amino acids can be removed using carboxypeptidase B. The tryptic cleavage can also be carried out by increasing the trypsin concentration or prolonging the reaction time such that cleavage additionally takes place at lysine(B29). In this case, a des(B30) insulin derivative results.
The insulin analog formed during the cleavage can be purified by standard chromatographic procedures (e.g. ion-exchange and reversed phase chromatography) and finally isolated by precipitation, crystallization or simple freeze-drying.
The precursor of the insulin analog preferably has the sequence
Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly lle Val Glu Gln Cys Cys Thr Ser lle Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
(SEQ ID NO: 7), for example the sequence of His(B0)-preproinsulin, or the sequence
Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg His Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly lle Val Glu Gln Cys Cys Thr Ser lle Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
(SEQ ID NO: 8), for example the sequence of His(B-1), Ala(B0) preproinsulin, or the sequence
Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg His Ala Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly lle Val Glu Gln Cys Cys Thr Ser lle Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
(SEQ ID NO: 9), for example the sequence of His(B-2), Ala(B-1), Ala(B0)-preproinsulin.
The present invention also relates to the abovementioned precursors of the insulin analogs according to the present invention, in particular the preproinsulins, the DNA sequences which code for a precursor of the insulin analog according to the present invention, the expression vehicles which contain a DNA sequence which codes for a novel precursor of the insulin analog according to the present invention, and a host cell which is transformed using such an expression vehicle.
The present invention furthermore relates to a pharmaceutical preparation comprising at least one insulin analog and/or at least one physiologically tolerable salt according to the present invention.
Preferably, the pharmaceutical preparation is distinguished in that it contains the insulin analog according to the invention and/or the physiologically tolerable salt thereof in dissolved, amorphous and/or crystalline form.
The pharmaceutical preparation alternatively furthermore contains a depot auxiliary, preferably protamine sulfate, the insulin analog and/or the physiologically tolerable salt thereof preferably being present in a cocrystallizate with the protamine sulfate.
The pharmaceutical preparation according to the present invention can alternatively additionally contain unmodified human insulin and/or a further insulin analog, preferably Gly(A21)-Arg(B31)-Arg(B32)-human insulin.
The present invention furthermore relates to an injectable solution having insulin activity, which contains the pharmaceutical preparation according to the present invention in dissolved form, preferably containing 1 xcexcg to 2 mg of zinc per ml, particularly preferably containing 5 xcexcg to 200 xcexcg of zinc per ml.
The present invention furthermore relates to the use of the insulin analog and/or its physiologically tolerable salt according to the present invention for the production of a pharmaceutical preparation which has an insulin activity having a delayed onset of action.
The object set at the outset is furthermore achieved by an insulin-zinc complex, comprising an insulin hexamer and 4 to 10 zinc ions per insulin hexamer, wherein the insulin hexamer consists of six molecules of an insulin analog of the formula I 
in which
(A1-A5) are the amino acid residues in the positions A1 to A5 of the A chain of human insulin or animal insulin,
(A15-A19) are the amino acid residues in the positions A15 to A19 of the A chain of human insulin or animal insulin,
(B8-B18) are the amino acid residues in the positions B8 to B18 of the B chain of human insulin or animal insulin,
(B20-B29) are the amino acid residues in the positions B20 to B29 of the B chain of human insulin or animal insulin,
R8 is Thr or Ala,
R9 is Ser or Gly,
R10 is lle or Val,
R14 is Tyr, His, Asp or Glu,
R21 is Asn, Asp, Gly, Ser, Thr, Ala, Glu or Gln,
R1 is any desired genetically encodable amino acid residue, absent or a hydrogen atom,
R2 is Val, Ala or Gly,
R3 is Asn, His, Glu or Asp,
R4 is Ala, Ser, Thr, Asn, Asp, Gln, Gly or Glu,
R30 is any desired genetically encodable amino acid residue or xe2x80x94OH,
Z is a hydrogen atom or a peptide residue having 1 to 4 genetically encodable amino acid residues, comprising 1 to 4 histidine residues (His).
The insulin-zinc complex preferably contains 5 to 8 zinc ions per insulin hexamer.
The insulin-zinc complex preferably contains an insulin hexamer which consists of six molecules of the insulin analog of the formula I described above according to the present invention.
The insulin-zinc complex according to the present invention is preferably also one wherein the B chain of the insulin analog of the formula I has the sequence
Phe Val His Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
(SEQ ID NO: 10), for example His(B3)-human insulin, or wherein the B chain of the insulin analog of the formula I has the sequence
Phe Val Asp Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
(SEQ ID NO: 11), for example Asp(B3)-human insulin.
The present invention also relates to a pharmaceutical preparation, comprising at least one insulin-zinc complex according to the invention and a pharmaceutical preparation comprising an acidic solution of at least one insulin analog and/or a physiologically tolerable salt thereof with an appropriate amount of zinc ions, which makes possible the formation of an insulin-zinc complex according to the present invention, the insulin analog and/or the physiologically tolerable salt preferably containing the insulin analog of the formula I according to the present invention described above or an insulin analog of the formula I whose B chain has the sequence with the number SEQ ID NO""s.: 3, 4, 5, 10 or 11.
The pharmaceutical preparation is preferably one which comprises the insulin-zinc complex in dissolved, amorphous and/or crystalline form.
The present invention also relates to an injectable solution having insulin activity, comprising the pharmaceutical preparation in dissolved form and preferably contains 1 xcexcg to 2 mg of zinc per ml, particularly preferably contains 5 xcexcg to 200xcexcg of zinc per ml.
The present invention also relates to the use of the insulin-zinc complex for the production of a pharmaceutical preparation which has an insulin activity having a delayed onset of action.
The insulin analogs according to the present invention are biologically active and exhibit a strongly delayed action after subcutaneous administration as a weakly acidic, clear solution containing 80 xcexcg of Zn++/ml (zinc/ml) in the dog. In the case of the insulin analog which is prolonged at the N-terminus of the B chain by histidine, His(B0), des(B30) human insulin (see SEQ ID NO.: 3), the profile of action depends, for example, very strongly on the amount of added zinc ions. A zinc-free preparation has no depot effect at all (total action 6-8 h, Example 8) and hardly differs in its pharmacodynamics from human insulin, while after addition of zinc ions (80 xcexcg/ml), a strong delay in action is found (total action approximately 16 h, Example 8). The observed depot effect is thus significantly more marked than that of NPH-insulin. Moreover, this analog has the advantage that the pharmacodynamics can be controlled by prespecification of the zinc content within a range which is not possible with human insulin. Formulations having a rapid onset of action can be prepared just like those having a moderately or strongly delayed action with an active substance just by varying the zinc content. Thus the profile of action can be individually adapted to the needs of the patient, either using a preparation having an appropriately preset zinc content or by mixing of preparations having a high and low zinc content by the physician or the patients themselves.
The analogs described here are furthermore those which, in comparison to human insulin, have an increased affinity for zinc ions.
In aqueous neutral solution, human insulin forms hexamers which in each case complex two zinc ions via the His(B10) side chains. These zinc ions cannot be removed by dialysis against aqueous buffers in neutral solution. Under the same conditions, the analogs described here bind more than 4 zinc ions. In the case of the His(B0)-des(B30)- and His(B3)-insulin according to the invention, these are approximately 7 zinc ions/hexamer; in the case of Asp(B3) insulin 4.2 zinc ions/hexamer were measured (Example 9).
It is known that in neutral solutions zinc leads to the formation of relatively high molecular weight associates and to the precipitation of the insulin. After the injection of a weakly acidic zinc-containing preparation which contains insulin which is dissolved to give a clear solution, the formation of insulin-zinc complexes and, as a result, the precipitation of the insulin occur in the subcutaneous tissue due to neutralization. Insulin goes into solution again from this depot and then passes into the blood stream and to the site of action with a delay. This delay in action is only slight in the case of human insulin, but strongly developed in the case of the analogs described here on account of the increased affinity for zinc. The increased zinc binding is therefore the basis of the zinc-dependent prolongation of action described above.
The present invention therefore not only relates to the insulin analogs described but also to the associated insulin-zinc complexes. These complexes differ from the corresponding human insulin-zinc complexes in that they have a higher content of firmly bound zinc. It is therefore evident that in addition to zinc other transition metal ions such as, for example, cobalt or copper can also be employed for the formation of corresponding complexes.